Rescuing HR DNA Repair in BRCA1-Mutation Carriers

Institution: Beckman Research Institute of the City of Hope
Investigator(s): Jeremy Stark, Ph.D. -
Award Cycle: 2012 (Cycle 18) Grant #: 18IB-0046 Award: $166,406
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2012)

This project was supported in part by a generous donation from the Katie Ann Buzbee Trust.

Carriers of mutations in BRCA1 show a significant increase in the lifetime risk of breast cancer (over 60%). To reduce cancer risk, the majority of patients identified as BRCA1 mutation carriers in North America undergo prophylactic mastectomy or oophorectomy. While such a preventative strategy is relatively effective at reducing cancer risk, some patients are reluctant to elect such surgery due to impacts on quality of life. An alternative prophylactic strategy could involve therapeutics that reduces the cancer predisposition resulting from BRCA1 deficiency. BRCA1 is important for the homologous recombination (HR) DNA repair pathway, which is critical for maintenance of genetic stability and tumor suppression. HR is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks.

We plan to identify specific molecular targets to suppress the HR defects caused by BRCA1 loss, with the long-term goal of developing prophylactic therapeutics to reduce the cancer risk of BRCA1 mutation carriers. Based on recently published and preliminary data, we postulate that disrupting either of two factors, 53BP1 (53BP1 was first described as a protein that interacts with p53) or RNF168 (a member of the E3 ubiquitin ligase cascade that lies “upstream” of 53BP1 in the DNA damage response pathway), can suppress the HR defects caused by BRCA1 loss.

We will examine HR using a series of assays for chromosomal break repair, as well as cytogenetic assays, based on mammalian cell culture model systems for BRCA1 deficiency. Our objective is to uncover functional domains of RNF168 and 53BP1 that are required to inhibit HR in BRCA1 deficient cells. Using this domain analysis, we propose to identify fragments of 53BP1 and RNF168 that can cause a dominant negative rescue of HR in BRCA1 deficient cells, which in future studies will be used for proof-of-principle experiments to develop this therapeutic approach. Furthermore, these mechanistic experiments are key to uncovering additional factors in the 53BP1/RNF168 signaling pathway. This represents an initial step to designing a therapeutic intervention to ”rescue” HR in BRCA1 deficient cells and ultimately in women that carry BRCA1 mutations.

Final Report (2014)

The long-term goal of this study is to improve therapeutic outcomes for patients with inherited mutations in the breast cancer susceptibility gene BRCA1. The BRCA1 protein promotes homologous recombination (HR), which is a cellular DNA repair process that is critical for maintaining genetic information. Accordingly, mutations in BRCA1 can cause inefficient HR, leading to accumulative loss of genetic information, which causes cancer. The intermediate-term goal of the project funded by this IDEA award is to define other genetic factors and pathways that influence the requirement for BRCA1 during HR. This research direction could lead to improved treatment of breast cancer. For example, factors that are important to inhibit HR in BRCA1 deficient cells are potential therapeutic targets to rescue HR in these cells. In addition, understanding how other factors affect the requirement for BRCA1 during HR, and hence tumor suppression, will provide insight into other genetic factors that may influence breast cancer risk for BRCA1 mutation carriers.

Accordingly, with support of this IDEA award, we sought to define specific factors that influence the requirement of BRCA1 during HR. For this, we applied molecular and cellular biology assays that enable a quantitative measure of the frequency of HR, using human cell culture model systems. Using this approach, we found that disrupting factors involved in the 53BP1/RNF168-signaling pathway can rescue HR in cells deficient for BRCA1. Namely, we find that disruption of either 53BP1 or RNF168 can suppress the HR defects caused by loss of BRCA1. Furthermore, we have made progress on defining the mechanism by which these factors influence HR. For one, we found that a peptide fragment of 53BP1 (dn53BP1) acts as a dominant-negative inhibitor that disrupts endogenous 53BP1 function. We also have identified regions of RNF168 important for its function to inhibit HR in BRCA1 deficient cells. We found that an N-terminal fragment of RNF168 (N221*) is proficient at inhibition of HR, and that conserved motifs within this fragment are important for this activity. As well, by examining other mutants of RNF168, we found that the ability for RNF168 to inhibit HR in BRCA1 deficient cells correlated with other cellular functions, including promoting the recruitment of 53BP1 to DNA damage.

Future plans are aimed at understanding the regulation of RNF168 function, and its genetic relationship to other factors that also inhibit HR. These mechanistic studies are an essential step in defining precise therapeutic targets to restore HR and reduce cancer risk in BRCA1 mutation carriers. As well, such mechanistic insight into this pathway will inform improved genomic-based approaches to predict breast cancer risk for individual BRCA1 mutation carriers.

An RNF168 fragment defective for focal accumulation at DNA damage is proficient for inhibition of homologous recombination in BRCA1 deficient cells. PMC4081061
Periodical:Nucleic Acids Research
Index Medicus: Nucleic Acids Res
Authors: Munoz MC, Yanez D, and Stark JM
Yr: 2014 Vol: 42 Nbr: 12 Abs: Pg:7720-33

RING finger nuclear factor RNF168 is important for defects in homologous recombination caused by loss of the breast cancer susceptibility factor BRCA1.
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Muñoz MC, Laulier C, Gunn A, Cheng A, Robbiani DF, Nussenzweig A, Stark JM
Yr: 2012 Vol: 287 Nbr: 48 Abs: Pg:40618-28